Taxonomic Applicability

Sex Applicability

Life Stage Applicability

Key Event Relationship Description

There is no direct information concerning insufficient or mis-repair of AFB1 promutagenic adducts leading directly to mutations in critical genes. It is well known, however, that in general when the repair of DNA adducts is either done incorrectly or is insufficient to remove the DNA adduct and correct the DNA sequence prior to DNA replication, a mutation at the site of the DNA adduct will result in the daughter cells upon DNA replication.

Evidence Supporting this KER

Biological Plausibility

Empirical Evidence

Evidence supporting the insufficient repair or mis-repair of pro-mutagenic DNA adducts is strong, but mainly indirect although there is some direct evidence from Archaea. The mechanism of DNA repair in Archaea is evolutionarily conserved and similar to that in eukaryotes. Banerjee et al. (2011) showed the P2 DNA polymerase in S. solfataricus bypasses the AFB1-N7-G adducts in an error-free manner but conducts error-prone replication past the AFB1-FAPy adduct and misinserts dATP, consistent with G:C to T:A transversion. (Banerjee et al., 2011).

Guo et al. (2005) used a transgenic yeast model expressing human CYP1A2 with defects in a range of DNA repair and cell cycle checkpoint pathways. This study demonstrated that nucleotide excision repair (NER), homologous recombination repair, post-replication repair, and several cell cycle checkpoint proteins all function in the repair of AFB1 adducts in yeast. Error-prone post-replication repair and apurinic endonucleases are secondary repair pathways that are used by cells when the major pathways are not functional or are overwhelmed. These secondary pathways tend to produce mutations.

The majority of both AFB1-N7-G and AFB1-N7-FAPy adducts are repaired by NER. (Alekseyev et al., 2004; Kew, 2013; Mulder et al., 2014). The evidence supporting the role of NER in humans is fairly strong. In populations in the Gambia and in China with high AFB1 exposure studies show that polymorphisms in the DNA repair gene x-ray repair cross complementary group (XRCC) are a risk factor for hepatocellular carcinoma (HCC). (Kirk et al., 2005; Long et al., 2006, 2008). In addition, these polymorphisms are associated with both increased levels of AFB1-DNA adducts and mutations at codon 249 of p53. (Long et al., 2008a; 2009). The correlation between AFB1 exposure, codon 249 mutations, and HCC has been established in humans. (Besaratinia et al., 2009) The secondary repair mechanisms play an important role in genesis of mutations from AFB1 adducts; this is particularly true for individuals with NER deficiencies such as those associated with XRCC polymorphisms. One possibility is that mutations occur through mis-repair by NER; alternatively, if NER is insufficient, the mutations may result through another pathway such as post-replication repair.

In the nematode, Caenorhabditis elegans, AFB1 is metabolized by CYP1, CYP2 and CYP3 family enzymes, and exposure to AFB1 results in significant DNA damage. (Leung et al., 2010). In C. elegans, whole genome sequencing was used to determine the mutational signature of AFB1 in worms deficient in DNA repair. A dose-response relationship was observed between AFB1 dose and G:C to T:A transversions and G:C to A:T transitions. (Meier et al., 2014). NER-deficient mutants showed an increase in these mutations compared to wild-type. In addition, the G:C to T:A transversions and G:C to A:T transitions were observed with a strand bias, indicative of transcription-coupled NER mechanisms similar to those observed in AFB1-associated p53 mutations in humans (Besaratinia et al., 2009; Carvalho et al., 2013; Gursoy-Yuzugullu et al., 2011; Symanska et al., 2009; Villar et al., 2011) This work with C. elegans is the first to link AFB1 adducts with mutations in a eukaryotic organism.

Other biological systems and datasets provide additional evidence from primarily mammalian species. The support provided by this evidence is based on the biological knowledge that a change in primary DNA sequence—in other words, a mutation—requires a mode of action that includes DNA damage. Mutations occur when DNA lesions are not repaired prior to cell replication (Preston and Williams, 2005; Pottenger et al., 2014). AFB1 forms two types of pro-mutagenic adducts: N7-AFB1-G and AFB1-FAPy DNA adducts, and resulting mutations are predominantly G:C to T:A transversions, which is the expected result of such adducts not being repaired or undergoing mis-repair (Bedard and Massey, 2006; Lin et al., 2014a,b).

These AFB1-induced DNA adducts undergo repair by SOS repair, NER, homologous recombination (HR), and post-replication repair (Bedard and Massey, 2006). NER appears to be most important for repair of AFB1-induced adducts in mammalian systems (Bedard and Massey, 2006). However, experiments in transgenic yeast expressing human CYP1A2 revealed different efficacy of the various DNA repair systems for AFB1-induced DNA adducts (Guo et al., 2005). Mutations were more likely to be induced in strains deficient in certain repair systems; mutations were also induced in strains with active secondary repair pathways, such as the pathways for error-prone post-replication repair or those relying on apurinic endonucleases.

The codon 249 of the p53 gene has been identified as a particular target of AFB1-induced adduction and subsequent mutation. Using DNA polymerase fingerprint analysis, Puisieux et al. (1991) showed specificity of the AFB1 epoxide binding for this codon.This specificity was not found with benzo[a]pyrene (B[a]P), although B[a]P also forms bulky, intercalating epoxides, (similar to AFB1 exo-epoxide), which result in bulky, intercalating N7-B[a]P-G adducts.

Uncertainties and Inconsistencies

Quantitative Understanding of the Linkage

In mammals, AFB1-N7-G and AFB1-N7-FAPy adducts are repaired by nucleotide excision repair (NER). AFB1-N7-G adducts are more readily repaired, or removed by spontaneous depurination, likely because these distort the structure of DNA to a greater extent than do AFB1-FAPy adducts. (Bedard and Massey, 2006). Species and tissue differences in repair capacities occur. Nuclear extracts from the livers of mice administered AFB1 in vivo seemed to repair AFB1 N7-G adducts at a greater rate than AFB1-N7-FAPy adducts, whereas repair of both types of adducts was similar in rat liver extracts. (Bedard et al., 2005). Given the heterogeneity in the human population with regard to DNA repair, the ability to repair AFB1 adducts could vary greatly.

There are no available data in either rodents or humans, which directly quantify the relationship between the number of AFB1 adducts and the frequency of mutation, either in surrogate genes or in the critical cancer gene. However, there are examples from other direct and indirect-acting alkylating agents (Doak et al., 2007; Gocke and Muller, 2009; Pottenger et al., 2009; Bryce et al., 2010; Dobo et al., 2011) that demonstrate non-linear dose-responses. Additional data on AFB1 could clarify the relationship between AFB1 pro-mutagenic adduct formation and induced mutations. This is a complex issue that requires further carefully designed experimental work.

There have been several studies that provide some quantitative information. Bailey et al. (1996) inserted an oligonucleotide containing a single AFB1-N7-G adduct into the genome of phage M13. In E. coli, the SOS response consists of the activation of over 20 unlinked genes involved in DNA damage tolerance and repair. (Smith and Walker, 1998). Replication of the oligonucleotide-bearing M13 phage in E. coli undergoing an induced SOS response yielded a mutation frequency of 4% for the AFB1-N7-G adduct (Bailey et al., 1996). The conditions in this study were extremely stringent, including single stranded DNA target (M13), and thus there was no ability to use the other DNA strand as an alternate template for bypass repair. These very stringent experimental conditions likely represent a significantly worst-case scenario for mutation induction, conditions with extremely limited repair opportunities and unlike normal conditions in eukaryotes.

While mutations at codon 249 of the p53 gene have been observed in association with HCC in humans, it is not known whether this mutation occurs as a direct result of adduct formation at this site early in the process or by some other mechanism. Human HepG2 hepatocytes were exposed to AFB1 and microsomes for metabolism. A dose-dependent increase in G:C to T:A transversions was observed at 10 additional locations as measured by ligation-mediated PCR, and at 4 additional locations using terminal-transferred dependent PCR. (Denissenko et al., 1998) These authors suggest that codon 249 may present an unusually mutagenic adduct conformation based on the local DNA sequence, and that a higher mutation rate may occur there rather than at other locations because of increased DNA polymerase bypass.

Leung et al. (2010) provide dose-response data for AFB1 resulting in DNA lesions in C. elegans, and Meier et al. (2014) provide dose-response data for AFB1-induced DNA base changes in C. elegans. However, the extent to which these quantitative relationships are applicable to humans or rodents is not yet clear.